OBJECTIVES:: Bacterial colonization is considered a major risk factor for necrotizing enterocolitis. The objective of this study was to test the hypothesis that Histamine-2 receptor blockers alter colonic bacterial colonization by analyzing and comparing the fecal microbiota in premature infants with and without Histamine-2 receptor blocker therapy using sensitive molecular biological techniques. METHOD:: Seventy-six premature infants ≤1500 grams or <34 weeks gestation were enrolled in this case-controlled, cross sectional study. Stool samples were collected from 25 infants receiving Histamine-2 receptor blockers and 51 babies who had never received them. Following DNA extraction and PCR amplification of 16S rRNA, 454 pyrosequencing was undertaken and the resulting sequences were subjected to comparison with published sequence libraries. RESULTS:: Proteobacteria and Firmicutes were the major phyla contributing to fecal microbial communities. Microbial diversity was lower, relative abundance of Proteobacteria (primarily of the family Enterobacteriaceae) was increased, while that of Firmicutes was decreased in the stools of infants receiving Histamine-2 receptor blockers compared with those who had never received them. CONCLUSIONS:: Although not designed to look specifically at the impact of Histamine-2 receptor blockers on the incidence of necrotizing enterocolitis, our study suggests that their use lowers fecal microbial diversity and shifts the microfloral pattern towards Proteobacteria. These alterations in fecal microbiota may predispose the vulnerable immature gut to necrotizing enterocolitis and suggest prudence in the use of Histamine-2 receptor blockers in the premature infant.